Background and Objectives: Breast tissue is dynamic and undergoes significant structural changes throughout a woman's life. The breast tissue architecture is maintained by a population of stem cells with self- renewal capacity which are essential for tissue repair and remodeling. Recently, the role of stem cells in breast carcinogenesis has been recognized as a high priority for translational breast cancer research. The stem cell hypothesis of breast carcinogenesis suggests that breast cancer development might be directly related to the size of the stem cell pool and its mitotic activity. Further, in the mammary gland stem cells are the only cell subpopulation that has capacity to accumulate all the oncogenic alterations. The proposed R01 project will fill the gaps in our understanding of the role of stem cells in breast carcinogenesis and their interplay with breast cancer risk factors and pathways. Specifically, using prospectively collected data and samples within Nurses' Health Study and Nurses' Health Study II cohorts, we will explore: 1) the prospective associations of parity, age at first birth, birthweight, and adolescent body size with CD44, CD24, and ALDH1A1 stem cell markers (n=1,348); 2) the association between stem cell markers in benign breast biopsies and subsequent breast cancer risk (~190 breast cancer cases/575 controls); and 3) the prospective associations of pre-biopsy circulating IGF-1 and IGFBP-3 (n= 472) with stem cell markers in subsequent benign biopsy samples. Methods: We will utilize an incident benign breast disease (BBD) study and a nested breast cancer case- control study within these cohorts to address the study aims. All BBD diagnoses are being confirmed with central pathology review. Some tissue microarrays (TMAs) from benign biopsies have been constructed in these cohorts, and additional TMAs will be added during the project period. Stem cell markers will be stained with commercially available antibodies using immunohistochemistry (IHC), and the staining results will be evaluated with automated image analysis. Information on plasma IGF-1 and IGFBP-3 is available from previous studies within these cohorts. Breast cancer risk factor data have been collected at baseline and updated biennially. Significance: We propose a highly novel investigation that will comprehensively examine the role of stem cell markers in breast carcinogenesis. The study aims to shed light on molecular pathways behind the observed associations of breast cancer risk factors with breast cancer risk as well as to identify markers that could advance future risk prediction in a large segment of high-risk women undergoing routine breast biopsies which could pave the way for novel personalized breast cancer prevention and surveillance strategies. The results will also add to the limited evidence on the association of IGF pathway with expression of breast stem cell markers. As stem cell activity is potentially modifiable via a variety of targeted therapies, the findings could translate into stem cell-directed pharmaceutical interventions aimed at breast cancer risk reduction in high-risk women with BBD in whom novel prevention strategies are urgently needed.